Reliable Detection of Deamidated Peptides from Lens Crystallin Proteins Using Changes in Reversed-Phase Elution Times and Parent Ion Masses

Abstract

Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (DeltaM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42% deamidation detection accuracy. Filtering SEQUEST identifications using RTS and DeltaM constraints resulted in>93% deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

“…Fig. 2 shows that the deamidated peptide DGEA-GAQ*GPPGPAGPAGER (COL1A1-0441-0 Van Doorn et al nomenclature (58)) can be distinguished by the MS ion envelope and elutes after the non-deamidated form (59). Interestingly, peptides derived from noncollagen proteins showed a very high to complete level of deamidation.…”

Detection, Evaluation and Minimization of Nonenzymatic Deamidation in Proteomic Sample Preparation

“…1D, an amidated peptide and its 13 C peak have the same retention time in RP chromatography, but a deamidated peptide has a different retention time than that of the corresponding amidated peptide. This has been used in the reliable identification of deamidated peptides (19,29). However, the method that we propose here is decidedly more convenient because it can be achieved easily by merely changing the parameters in database searches.…”

Lens β-crystallins: The role of deamidation and related modifications in aging and cataract

“…Conversion of an amide to a carboxylic acid causes a 0.984 Da mass increase, which is almost identical to the mass of the non-deamidated peptide having an extra neutron from naturally occurring heavy isotopes of the constitute elements. Since mass spectrometers can accurately measure the monoistopic masses of peptides, it is easy to distinguish between non-deamidated and deamidated forms if the resulting chromatographic peaks of the two peptides forms are chromatographically resolved from one another (Dasari et al, 2007). Unfortunately, this is not always possible.…”